Advanced Genetics

Advanced Genetics

Why Study Genetics?

Why Study Genetics?

1. Historical and aesthetic appreciation

Why Study Genetics?

1.  Historical and aesthetic appreciation

2.  Practical applications

- Recognize genetic experiments

Why Study Genetics?

1.  Historical and Aesthetic appreciation

2.  Practical applications

-  Recognize genetic experiments

-  Interpret genetic experiments
Why Study Genetics?

1.  Historical and Aesthetic appreciation

2.  Practical applications

-  Recognize genetic experiments

-  Interpret genetic experiments
-  Conduct genetic experiments
 What is a
genetic experiment?
Classification of Experiments

1. Observational

2. Interventional
Classification of Experiments

1.  Observational

-  Visual (microscopy)

-  Physiology (blood pressure)

-  Chemical analysis (serum glucose)

-  Biochemical analysis (sequence

genome, proteome, metabalome, etc.)
Classification of Experiments
Interventional
1. Environmental
- pharmacology
- nutrition
- temperature
Classification of Experiments
Interventional
1. Environmental
- pharmacology
- nutrition
- temperature

2. Mechanical
- biochemistry
- physiology
- surgical (transplantation)
Classification of Experiments
Interventional
1. Environmental

2. Mechanical

3. Genetic
- manipulate genes

Interventional experiments are powerful

because cause/effect relationships can be
inferred
Classification of Experiments
Interventional Genetic Experiments
1. Manipulate genes

2. Observe phenotype

3. Conclusion

Interventional experiments are powerful

because cause/effect relationships can be
inferred
 Cause

How does an experiment establish cause?

Why is it important to establish cause?

 Cause
Observation 1 Observation 2
 Cause
Observation 1 Observation 2

Correlation
 Cause
Observation 1 Observation 2

Correlation
 Cause
Experiment
Intervention 1 Observation 1

Control
No Intervention Observation 1

Positive Conclusion: Intervention causes change

Negative Conclusion: Intervention does not cause change
 Cause
Experiment
Intervention 1 Observation 1 Observation 2

Control
No Intervention Observation 1 Observation 2

Positive Conclusion: Intervention causes change of 1 and 2

What conclusion about relationship of 1 and 2?

 Cause
Experiment
Intervention 1 Observation 1 Observation 2

Control
No Intervention Observation 1 Observation 2

Positive Conclusion: Intervention causes change of 1 and 2

What conclusion about relationship of 1 and 2?

CORRELATION
 Cause
Intervention 1 JUSTIFIED

Observation 1 Observation 2

Intervention 1

Observation 1 NOT JUSTIFIED

Observation 2
Categories of genetic experiments
1.  Method

- Classical (mutagens and breeding)

- Molecular (Molecular biology)

2. Logic

- Forward (phenotype to gene)

- Reverse (gene to phenotype)
Example: Mendel’s peas
Found mutant pea strains – wrinkled and
smooth.

Bred to generate heterozygotes.

Observed 3:1 segregation ratio.

Conclusion regarding dominant and recessive

alleles.
Example: Transcription factor binding
Perform ChIP-seq with histone mark.

Perform ChIP-seq with transcription factor.

Identify transcription factor binding sites that have the

histone mark.

Draw conclusion regarding how histone mark affect

transcription factor binding.
Example: Protein crystallography
Express wild-type protein from plasmid in bacteria,
purify, crystallize and solve 3-D structure.

Perform site directed mutagenesis on plasmid.

Express mutant protein from plasmid in bacteria, purify,

crystallize and solve 3-D structure.

Draw conclusion regarding residue and structure.

Example: Dominant negative protein
Design mutant ERK kinase protein that interferes with
function of wild-type protein.

Express in cells from plasmid, measure signal

transduction.

Draw conclusion regarding role of ERK in signaling.

Example: Dominant negative protein
Design mutant ERK kinase protein that interferes with
function of wild-type protein.

Synthesize protein with a machine.

Inject protein into cells, measure signal transduction.

Draw conclusion regarding role of ERK in signaling.

Proposition: The essence of genetics is the
analysis of the relationship between
GENOTYPE and PHENOTYPE

Geneticists primarily:
1.  Manipulate genotype (Intervention)

2.  Analyze phenotype

(Observation)

3. Draw conclusions
 Genotype
1. Definition: All the genetic information contained in an organism; the genetic
constitution of an organism with respect to one or a few genes under consideration.

2. Distinctions:
A.  One gene vs. Entire genome

B.  Genetic vs. Molecular

Alleles known by Alleles known by DNA

phenotype they cause sequence

Allele Phenotype DNA

unc-1(+) wild type wild type
unc-1(e1) mild uncoordinated missense codon
unc-1(e2) severe uncoordinated deletion

C. Wild type is by definition

D. Complete description
- genetic: wild type or mutant at every locus
- molecular: sequence of the genome
 Phenotype
1.  Definition: The observable characteristics of an
individual, which are a result of genotype and
environment.

2.  Common ways to determine phenotype:

- visual inspection, including microscopy, staining, etc.

- biochemical assay
- chemical assay
- behavioral assay
 Caenorhabditis elegans

Brenner,  Sidney  (1974).  The  gene5cs  

 of  Caenorhabdi+s  elegans.  Gene5cs  77:  71-­‐94  
 
(9,310  cita5ons,  2002  Nobel  prize)  
 What makes an organism
suitable for genetic analysis?
1.  Ease  of  culture.  

2.  Reproduc5ve  system  (self  fer5le  

hermaphrodites  and  males).  

3.  Small  genome  and  limited  number  of  

chromosomes.  
 Introduction/Rationale
1.  “How  genes  might  specify  the  complex  structures  
found  in  higher  organisms  is  a  major  unsolved  problem  
in  biology”  
2.  Necessary  to  find  mutants  (genotype)  and  analyze  the  
structure  of  the  nervous  system  (phenotype).  
3.  “Some  eight  years  ago,  when  I  embarked  on  this  
problem,  I  decided  that  what  was  needed  was  an  
experimental  organism  which  was  suitable  for  
gene5cal  study  and  in  which  one  could  determine  the  
complete  structure  of  the  nervous  system.  Drosophila,  
with  about  105  neurons,  is  much  too  large,  and,  
looking  for  a  simpler  organism,  my  choice  eventually  
seWled  on  the  small  nematode,  Caenorhabdi+s  
elegans.”  
 Properties of C. elegans
   -­‐  Self-­‐fer5lizing  hermaphrodite:    XX  
   Male:                      XO  

 -­‐  1  mm  length  

 
 -­‐  3.5  day  life  cycle  
 
 -­‐  Small,  possibly  fixed  
     number  of  cells  
 Results
 1.    Isolate  muta5ons  
 
 -­‐  Mutagenize  with  chemical  mutagen  
 
 -­‐  Screen  for  mutant  animals  
 Results
 2.    Posi5on  in  the  genome  (mapping)  
 
 A.    Autosomal  vs.  sex  linked  
 
 B.  Two-­‐factor  mapping  (Distance)  
Measure  recombina5on  frequency  between  two  muta5ons  
       a  b  
       +  +  
 
   
 Results
 2.    Posi5on  in  the  genome  (mapping)  
 
 A.    Autosomal  vs.  sex  linked  
 
 B.  Two-­‐factor  mapping  (Distance)  
Measure  recombina5on  frequency  between  two  muta5ons  
       a  b  
       +  +  
 
 C.  Three-­‐factor  mapping  (Order)  
 
 +  a  b          a  +  b  
 c  +  +          +  c  +  
 Results
 3.    Complementa5on  
 
   -­‐  Purpose,  determine  if  two  muta5ons  affect  the  
     same  gene  
 
               m1  
               m2    
 Results
 Table  4  
 
1.  Linkage  to  par5cular  autosome  or  X  chromosome  
2.  Complementa5on  
 Results
 Figure  3  and  maps  
 
1.  2-­‐factor  mapping  
2.  3-­‐factor  mapping  
 Discussion/Conclusions
 Brenner  developed  methods  for  manipula5ng  the  
genotype  of  C.  elegans,  making  gene5c  analysis  possible.  
 
1.  Isola5on  of  muta5ons  
2.  Mapping  
 -­‐    Linkage  to  chromosome  
 -­‐  Posi5on  on  a  chromosome  
     -­‐  2-­‐factor  
 -­‐  3-­‐factor  
3.  Complementa5on  tests  
 
These  techniques  made  it  possible  to  address  
biological  ques5ons.